CN108872168B - Rapid quantitative detection method of vitamin D using fluorimetry in lateral flow cartridge - Google Patents

Rapid quantitative detection method of vitamin D using fluorimetry in lateral flow cartridge Download PDF

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CN108872168B
CN108872168B CN201810366783.3A CN201810366783A CN108872168B CN 108872168 B CN108872168 B CN 108872168B CN 201810366783 A CN201810366783 A CN 201810366783A CN 108872168 B CN108872168 B CN 108872168B
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hydroxyvitamin
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崔义烈
朱厚暾
金禛洙
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Boditech Med Inc
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Abstract

The present invention relates to a method for rapid quantitative determination of vitamin D by fluorometry in a lateral flow cartridge, which does not require the conventional time-and labor-intensive sample pretreatment step and thus enables rapid and accurate vitamin D determination.

Description

Rapid quantitative detection method of vitamin D using fluorimetry in lateral flow cartridge
Technical Field
The present invention relates to a Rapid quantitative vitamin D detection method (Rapid quantitative immunoassay method for vitamin D using a lateral flow cartridge) using a fluorometry method in a lateral flow cartridge.
Background
Vitamin D is a fat-soluble vitamin that helps the bone to grow healthily and maintain the bone healthy by regulating the values of calcium and phosphorus, and also functions to regulate the immune system and blood glucose and suppress the occurrence of various infections. If vitamin D is insufficient, the incidence of osteoporosis, fall injuries, and hip fractures is high, and the incidence of various cancers or autoimmune diseases is also increased.
Vitamin D (calciferol) is classified as D2(ergocalciferol) and D3(cholecalciferol ). Vitamin D2Prepared from yeast and ergosterol (ergosterol) as phytosterol (sterol), vitamin D3It is known that it can be produced from 7-dehydrocholesterol (7-dehydrocholestrol) which is a precursor of cholesterol when the skin is irradiated with sunlight ultraviolet rays, and that their effectiveness is almost the same.
Vitamin D, whether synthesized in vivo or ingested from food, is in the form of prohormone (pro-hormone) which is a precursor of active hormones, and needs to be converted into an activated form in the liver and kidney to exert biological functions. In the liver, vitamin D3And vitamin D2Are respectively metabolized into 25-hydroxy vitamin D by hydrogen oxidation at the 25 carbon position3(25-OH-D3) And 25-hydroxyvitamin D2(25-OH-D2) The above 25-hydroxyvitamin D3And 25-hydroxyvitamin D2Is transported toKidney, carbon position No. 1 is again oxidized by hydrogen, thus 25-hydroxy vitamin D3Is metabolized into the active hormone 1, 25-dihydroxy vitamin D325-hydroxy vitamin D2Is metabolized into the active hormone 1, 25-dihydroxy vitamin D2。
For monitoring and controlling vitamin D deficiency or excess, the most suitable assay is the determination of 25-hydroxyvitamin D, which is required for the determination of an accurate amount of 25-hydroxyvitamin D3(25-OH-D3) And 25-hydroxyvitamin D2(25-OH-D2) All measurements were performed.
As a detection method of vitamin D which is generally used, there are roughly an LC-MS/MS (Liquid Chromatography-Mass Spectrometry/Mass Spectrometry) method and a chemiluminescence immunoassay (CLIA). The LC-MS/MS method is a standard detection method of vitamin D, and has the advantages of high accuracy, high sensitivity and high precision, and can also detect vitamin D3And D2The measurement is distinguished. However, the LC-MS/MS method has a disadvantage in that expensive analysis equipment is required. In addition, the CLIA method has advantages in that rapid measurement can be achieved due to the use of automated equipment, and also in that sensitivity and accuracy are high and a pretreatment process is not required. However, the CLIA method has a disadvantage in that the results may vary from detection authority to detection authority because the CLIA method varies greatly depending on the reagent or method.
On the other hand, Korean laid-open patent No. 2015-0100935 discloses "a method and a kit for measurement of vitamin D", characterized in that a sample is treated with a surfactant having a steroid skeleton. Further, Korean laid-open patent No. 2015-0110680 discloses "a method and kit for detecting 1, 25-dihydroxyvitamin D and its related antibodies", but does not disclose the rapid quantitative detection method of vitamin D using a fluorimetric assay in a lateral flow cartridge of the present invention.
Disclosure of Invention
The present invention has been made in view of the above-mentioned needs, and the present inventors have developed a method capable of rapidly quantifying the concentration of vitamin D present in blood by an immunoassay using an anti-25-hydroxyvitamin D antibody and a lateral flow and fluorometry method, and have optimized temperature conditions so as to be able to accurately distinguish a fluorescence value that varies depending on the amount of vitamin D in a sample, thereby completing the present invention.
In order to solve the above problems, the present invention provides a method for rapid quantification of vitamin D using fluorometry in a lateral flow cartridge, comprising: (a) mixing a biological sample and a releasing buffer solution, and reacting at 32-37 ℃ for 3-7 minutes to prepare a first reactant; (b) adding a detection buffer solution to the first reactant obtained in the step (a) and reacting the mixture at 32 to 37 ℃ for 13 to 17 minutes to prepare a second reactant; and (c) loading the second reactant obtained in the step (b) into a lateral flow tube, reacting for 6-10 minutes, and measuring the fluorescence intensity by using a fluorescence measuring instrument.
The present invention relates to a method for quantifying vitamin D in a biological sample using a lateral flow cartridge and fluorometry, which does not require the conventional time and labor intensive sample pretreatment step and thus enables rapid and accurate vitamin D measurement.
Drawings
FIG. 1 is a schematic diagram showing the principle of lateral flow reaction and the principle of fluorescence measurement used in the vitamin D quantification method of the present invention.
FIG. 2 is a graph analyzing the results of fluorescence measurements according to different vitamin D concentrations under various temperature conditions. A T region: fluorescence value of detection line (test line), C region: fluorescence value of control line (control line).
FIG. 3 is a graph showing the results of measurements using the method and apparatus of the present invention using 45 serum samples and the results of quantitative analysis using a control apparatus (Cobas E411). Y: measurement of reference device Cobas, X: the measured value of the present invention.
Detailed Description
In order to achieve the object of the present invention, the present invention provides a method for rapidly quantifying vitamin D by fluorimetry using a lateral flow cartridge in a biological sample.
The rapid vitamin D quantification method of the present invention uses an immunoassay method using a competitive reaction, a fluorescence-labeled detection antibody (anti-25-hydroxyvitamin D antibody) present in a detection buffer, and vitamin D (25-hydroxyvitamin D; 25(OH) D) present in a sample (biological sample)2And 25(OH) D3) Forming an antigen-antibody complex, allowing the remaining detection antibody not forming the complex to pass through the nitrocellulose medium of the lateral flow cartridge strip while moving, and allowing the detection antibody to be immobilized on a nitrocellulose developing membrane
Figure BDA0001637394700000031
Figure BDA0001637394700000032
The competitor (BSA-25(OH) D complex) is bound, and fluorescence emitted from the detection antibody bound to the competitor is measured with a fluorometer (ichroma (TM) Reader, Boditech Med).
The term "immunoassay" refers to a method for measuring a target substance in a trace amount from a sample containing many impurities by utilizing the specific binding ability between an antigen and an antibody. When a labeling method using a fluorescent (or luminescent) substance, a radioactive compound, an enzyme, a metal, or the like is combined with an immunoassay method, the measurement sensitivity is dramatically improved.
The method for rapid quantification of vitamin D according to the present invention may specifically include, but is not limited to:
(a) mixing a biological sample and a releasing buffer solution, and reacting at 32-37 ℃ for 3-7 minutes to prepare a first reactant;
(b) adding a detection buffer to the first reactant in the step (a) and reacting the mixture at 32 to 37 ℃ for 13 to 17 minutes to produce a second reactant; and
(c) and (c) loading the second reactant obtained in the step (b) into a lateral flow tube, reacting for 6-10 minutes, and measuring the fluorescence intensity by using a fluorescence measuring instrument.
In the method for rapidly quantifying vitamin D according to one embodiment of the present invention, the vitamin D may preferably be 25-hydroxyvitamin D3(25-OH-D3) And 25-hydroxyvitamin D2(25-OH-D2) But is not limited thereto.
In the past decades, when the concentration of vitamin D in vivo is measured, 1,25(OH) D, which is a completely active form of vitamin D, is measured, but since 25(OH) D in liver or blood becomes 1,25(OH) D in kidney, it is judged that the measurement of 1,25(OH) D does not reflect the present vitamin reserves in vivo but reflects the function of kidney, and recently, the concentration of 25(OH) D in blood is measured when the concentration of vitamin D in vivo is measured.
In the method for rapid quantification of vitamin D according to an embodiment of the present invention, the release buffer in step (a) may include 0.4 to 0.6N sodium hydroxide and 35 to 45% (v/v) DMSO (dimethyl sulfoxide), but is not limited thereto, and the release buffer may be used to release 25-hydroxyvitamin D in a biological sample from vitamin D binding protein (vitamin D binding protein).
In the present invention, the term "biological sample" refers to a liquid phase or a fluid substance similar to a liquid, and examples thereof include blood, saliva, urine, sweat, interstitial or intracellular body fluid, and substances extracted therefrom, and the blood may include whole blood, plasma, serum, or blood, plasma, serum, and the like subjected to a predetermined treatment (for example, anticoagulation), but is not limited thereto, and the biological sample may be used in a manipulated or unprocessed state.
In the method for rapid quantification of vitamin D according to an embodiment of the present invention, the detection buffer in step (b) may include a fluorescently labeled anti-25-hydroxyvitamin D antibody, specifically, a fluorescently labeled anti-25-hydroxyvitamin D antibody, a fluorescently labeled anti-rabbit IgG, 0.3 to 0.7% (w/v) gelatin, 100 to 200mM sodium chloride, 0.05 to 0.15% (w/v) sodium azide, 0.3 to 0.55% (w/v) CHAPS (3- [ (3-Cholamidopropyl) dimethylammonio ] -1-propanesulfonate hydrate), and 400 to 600mM Tris-HCl, but not limited thereto.
Anti-25-hydroxy group in the above detection bufferVitamin D antibodies and 25-hydroxyvitamin D2And 25-hydroxyvitamin D3Any of them can be bound to the antibody, and commercially available antibodies can be purchased and used.
In the method for rapid quantification of vitamin D according to an embodiment of the present invention, the lateral flow cartridge is in the form of a test strip, and may include: a sample injection unit for injecting a sample; a test line (test line) in which a complex of Bovine Serum Albumin (BSA) and 25-hydroxyvitamin D is immobilized at a predetermined distance from the sample injection unit; control line (control line) immobilized with rabbit IgG; but is not limited thereto.
In addition, the method for rapid quantification of vitamin D according to the present invention is characterized in that the fluorescence intensity is inversely proportional to the concentration of vitamin D in the biological sample, because the anti-25-hydroxyvitamin D antibody in free (free) form, which does not form an antigen-antibody complex, among the fluorescently labeled anti-25-hydroxyvitamin D antibodies present in the detection buffer is captured by the BSA-25-hydroxyvitamin D complex immobilized on the detection line, and the amount of fluorescence emitted therefrom is measured. That is, if 25-hydroxyvitamin D is present in a large amount in blood, the anti-25-hydroxyvitamin D antibody mostly forms an antigen-antibody complex, while the amount of the anti-25-hydroxyvitamin D antibody in a free form decreases, so that the amount of the anti-25-hydroxyvitamin D antibody bound to the BSA-25-hydroxyvitamin D complex immobilized on the detection line of the lateral flow cartridge is small, and the final fluorescence signal intensity is recognized to be weak, whereas if 25-hydroxyvitamin D is present in a small amount in blood, the final fluorescence signal intensity is recognized to be strong.
Another fluorescently labeled anti-rabbit IgG in the detection buffer was used as an internal control (internal control) and bound to rabbit IgG immobilized on a control line in a lateral flow tube, showing fluorescence. Therefore, if the reaction is a normal reaction, a certain amount of fluorescence is exhibited in all reactions.
In the present invention, a fluorescent substance is used as a label for detecting an antigen-antibody complex, but the present invention is not limited thereto, and an enzyme, a ligand, a luminescent substance, a microparticle, a radioisotope, or the like can be used.
Fluorescent substances used as detection markers include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, Eu3+、Eu3+Chelates or cryptates, etc.; the enzyme includes acetylcholinesterase, alkaline phosphatase, beta-D-galactosidase, horseradish peroxidase, beta-lactamase, etc.; as the ligand, biotin derivatives and the like are included; as the light-emitting substance, acridinium ester, isoluminol derivative, or the like is included; as the fine particles, colloidal gold, colored latex, etc. are included; as the radioactive isotope may be included57Co、3H、125I、125I-Bonton Hunter reagent, and the like, but is not limited thereto.
The range in which the method of the present invention can quantify vitamin D present in a biological sample is 8.0 to 70 ng/mL.
The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
Materials and methods
1. Reagent and apparatus
The fluorescently labeled anti-25 (OH) D antibody and anti-rabbit IgG antibody were prepared by purchasing and conjugating (conjugation) the respective anti-25 (OH) D antibody and anti-rabbit IgG antibody and FPR-648. Bovine serum albumin (bovine serum albumin) and 25-hydroxyvitamin D to be immobilized on the detection line3(25(OH)D3) Or prepared for bonding after purchase separately.
The release buffer (discharging buffer) was made with a composition of 0.5N sodium hydroxide and 40% DMSO, and the detection buffer (detection buffer) was made with 0.5% gelatin, 150mM sodium chloride, 0.1% sodium azide (NaN)3) 0.4% CHAPS, 500mM Tris-HCl (pH6.8), a fluorescently labeled anti-25 (OH) D antibody and a fluorescently labeled anti-rabbit IgG.
2. Concentration calculation of vitamin D by fluorescence value
The ratio (ratio) is the value obtained by dividing the amount of fluorescence (T-area) measured in the detection line by the amount of fluorescence (C-area) measured in the control line.
Using standard equipment, a calibrator (calibrator) for the concentration conditions (0, 5, 10, 20, 30, 50, 70, 100ng/ml) of the 8 intervals that have been measured was prepared, and the ratio (ratio) value under each concentration condition was measured and determined with a fluorescence measuring instrument (ichroma. TM. Reader, Boditech Med). A calibration curve (calibration curve) is created based on this and the ratio (ratio) obtained when an unknown sample is measured is substituted into the calibration curve, whereby the concentration of vitamin D in the sample can be calculated.
Example 1 analysis of variation of results values based on temperature conditions
When the concentration of vitamin D in the sample was measured, the influence of temperature under the reaction conditions was evaluated. After mixing 50. mu.l of the release buffer and 50. mu.l of vitamin D samples at concentrations of 0, 10, 30 and 70ng/ml, respectively, the mixture was reacted at 20, 22, 25, 30, 32, 35, 37 or 39 ℃ for 5 minutes. Then, 100. mu.l of detection buffer was added to the reaction mixture, and the reaction was carried out at the above-mentioned 8 temperatures for 15 minutes. The reaction product was loaded in an amount of 75. mu.l into the sample injection part of the cartridge set in the slot of the fluorescence measuring instrument, and the fluorescence intensities of the control line and the detection line were measured after 8 minutes from closing the slot of the fluorescence measuring instrument.
As a result, as shown in fig. 2, it was confirmed that the fluorescence amount (C area) of the control line showed a constant value in all cases regardless of the sample concentration and the temperature condition. However, it was confirmed that the fluorescence amounts (T area) of the detection lines for vitamin D at concentrations of 0 and 10ng/ml could not be distinguished under the temperature conditions of 20, 22, 25 and 30 ℃. In addition, at 39 ℃, there was a tendency that the difference in the fluorescence measurement values between different concentrations was again reduced compared to the conditions at 32, 35 and 37 ℃. When the ratio (ratio) obtained by dividing the fluorescence amount (T-area) measured in the detection line by the fluorescence amount (C-area) measured in the control line and the percentage conversion value thereof were observed, the fluorescence measurement value based on the vitamin D sample concentration showed a significant difference, and it was confirmed that the temperature condition more favorable for the differentiation of the sample concentrations was in the range of 32 to 37 ℃.
Example 2 Performance analysis Using Standard conditions
From example 1 above, it was determined that the method of the present invention was performed at 35 ℃, and the accuracy and precision of sample analysis using the method of the present invention was analyzed.
Accuracy the assay results for the standard substances were analyzed in terms of manufacturing lot (Between-lot), subject (Between-person), number of repetitions/days of the same lot (Between-day), and place of execution of the same lot (Between-site).
[ TABLE 1 ]
Accuracy analysis
Figure BDA0001637394700000081
Coefficient of variation: percentage of standard deviation (%). relative to mean
As a result, it was confirmed that the measurement results of different batches, different subjects, different repeat days, and different sites were not greatly different under the various conditions as shown in table 1. Further, as shown in table 2 below, the results of repeating the measurement 10 times for 3 concentrations of the standard substance using 3 batches of the product confirmed that the recovery rate was at a level of 97 to 100%, and the measurement was the same value as the concentration of the standard substance. From this, it was found that the vitamin D measurement method of the present invention is excellent in accuracy.
[ TABLE 2 ]
Accuracy analysis
Figure BDA0001637394700000091
Recovery rate: mean/standard substance concentration of measured values X100
Example 3 comparison of correlation
The concentration of vitamin D was measured for 45 serum samples using the fluorescence measuring device of the present invention and Cobas e 411 (roche, switzerland) as an immunochemical analyzing device, and comparative analysis thereof was performed. As a result, as shown in fig. 3, the quantitative analysis result values of both devices showed a linear regression formula of Y — 1.1629X-0.2829, and the correlation coefficient R showed 0.9551. It was found that the measured values of the present invention were not significantly different from the measured values of the reference device.

Claims (4)

1. A method for rapid quantification of vitamin D using fluorimetry in a lateral flow cartridge, comprising:
(a) mixing a biological sample and a release buffer solution, and reacting at 32-37 ℃ for 3-7 minutes to prepare a first reactant;
(b) adding a detection buffer solution into the first reactant in the step (a) and reacting at 32-37 ℃ for 13-17 minutes to prepare a second reactant; and
(c) loading the second reactant obtained in the step (b) into a lateral flow tube, reacting for 6-10 minutes, and measuring the fluorescence intensity by using a fluorescence measuring instrument;
the release buffer solution in the step (a) comprises 0.4-0.6N sodium hydroxide and 35-45% (v/v) dimethyl sulfoxide (DMSO).
2. The method for rapid quantification of vitamin D using fluorimetry in a lateral flow cartridge according to claim 1, characterized in that said vitamin D is 25-hydroxyvitamin D, 25-OH-D.
3. The method of claim 1, wherein the detection buffer of step (b) comprises a fluorescently labeled anti-25-hydroxyvitamin D antibody.
4. The method of claim 1, wherein the lateral flow cartridge of step (c) is in the form of a test strip and comprises, in order: a sample injection unit for injecting a sample; a detection line which is located at a position spaced apart from the sample injection part by a predetermined distance and to which a complex of bovine serum albumin and 25-hydroxyvitamin D is fixed; control line, immobilized rabbit IgG.
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